The Pathway to Activation of the Vav Proto-Oncogene

描述（由申请人提供）：原癌蛋白Vav是淋巴细胞信号转导途径的关键调节剂，是Rho GTPases的鸟嘌呤核苷酸交换因子（GEF）。Vav的催化DH结构域被邻近的结合GEF活性位点的n端肽自抑制。Lck磷酸化该臂中一个隐藏的酪氨酸，使该臂与DH结构域分离，激活Vav。本研究结合生物化学和核磁共振光谱研究来发现Vav活化的途径。我们假设A) Vav n端额外的酪氨酸基序（y基序）可能结合DH结构域并有助于自身抑制，B)暴露的y基序的初始磷酸化通过其SH2结构域招募Lck，使动力学上不利的磷酸化事件以分子内方式发生。磷酸化的Vav蛋白对Lck SH2结构域的亲和性，以及Vav n端肽对DH结构域的亲和性将被确定，揭示Vav n端哪些y基序可以结合DH结构域，并在磷酸化时招募Lck。磷酸化和SH2结合对dh抑制肽亲和力和结合动力学的影响将被确定，揭示这些初始步骤是否有助于缓解自身抑制。将确定Lck激酶结构域催化的Vav肽磷酸化的动力学参数，包括游离的和分子内结合到DH结构域的。还将确定全长Lck的参数，揭示sh2介导的磷酸化位点对接在多大程度上可以克服DH结构域对隐藏酪氨酸的隔离作用。通过多场核磁共振弛豫色散分析，测量自抑制Vav中的酰胺和甲基[mu]s-ms时间尺度动力学。这些数据将量化这些运动的速率，以及基态和激发态的种群以及它们之间的化学位移差异。化学位移和速率数据，结合生化测量的螺旋离解速率，将揭示核磁共振动力学是否代表了螺旋离解状态的偏移。核磁共振动力学速率与磷酸化动力学的比较将揭示核磁共振动力学是否可以代表控制Lck激活Vav速率的运动。这些结果将验证我们的想法，即自抑制蛋白通常具有暴露的“访问点”，以克服其设计中固有的激活动力学障碍。

DESCRIPTION (provided by applicant): The protooncoprotein Vav, a key regulator of lymphocyte signal transduction pathways, is a guanine nucleotide exchange factor (GEF) for Rho GTPases. The catalytic DH domain of Vav is autoinhibited by an adjacent N-terminal peptide, which binds the GEF active site. Phosphorylation of a buried tyrosine in this arm by Lck dissociates the arm from the DH domain, activating Vav. This proposal integrates biochemical and NMR spectroscopic investigations to discover the pathway to Vav activation. We hypothesize that A) additional tyrosine motifs (Y-motifs) in the Vav N-terminus may bind the DH domain and contribute to autoinhibition, and B) initial phosphorylation of exposed Y-motifs recruits Lck through its SH2 domain, enabling the kinetically disfavored phosphorylation event to occur in intramolecular fashion. Affinities of phosphorylated Vav proteins for the Lck SH2 domain, and of Vav N-terminal peptides for the DH domain will be determined, revealing which Y-motifs in the Vav N-terminus can bind the DH domain, and, when phosphorylated, recruit Lck. Effects of phosphorylation and SH2 binding on DH-inhibitory peptide affinity and binding kinetics will be determined, revealing whether these initial steps could contribute to relief of autoinhibition. Kinetic parameters for Lck kinase domain-catalyzed phosphorylation of Vav peptides, both free and bound intramolecularly to the DH domain, will be determined. Parameters will also be determined for full-length Lck, revealing the extent to which SH2-mediated docking to phosphorylated sites can overcome the sequestering effect of the DH domain toward the buried tyrosine. Amide and methyl group [mu]s-ms timescale dynamics in autoinhibited Vav will be measured by NMR relaxation dispersion analysis at multiple fields. The data will quantitate the rates of these motions, as well as the populations of the ground and excited states and the chemical shift differences between them. The chemical shift and rate data, combined with biochemically-measured helix dissociation rates, will reveal if the NMR dynamics represent excursions to a helix-dissociated state. Comparison of NMR dynamics rates with kinetics of phosphorylation will reveal if NMR dynamics could represent motions that govern the rate of Vav activation by Lck. These results will test our ideas that autoinhibited proteins often possess exposed "access points" to overcome kinetic barriers to activation inherent in their design.